Aav-mediated gene therapy for rpgr x-linked retinal degeneration

ABSTRACT

Described herein are methods of preventing, arresting progression of or ameliorating vision loss and other conditions associated with retinitis pigmentosa and x-linked retinitis pigmentosa in a subject. The methods include administering to said subject an effective concentration of a composition comprising a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding a normal retinitis pigmentosa GTPase regulator (RPGR gene), or fragment thereof, under the control of regulatory sequences which express the product of the gene in the photoreceptor cells of the subject, and a pharmaceutically acceptable carrier.

BACKGROUND OF THE INVENTION

Photoreceptors function cooperatively with the retinal pigmentepithelium (RPE) to optimize photon catch and generate signals that aretransmitted to higher vision centers and perceived as a visual image.Disruption of the visual process in the retinal photoreceptors canresult in blindness. Genetic defects in the retina cause substantialnumbers of sight-impairing disorders by a multitude of mechanisms.

Among photoreceptor dystrophics, the X-linked forms of retinitispigmentosa (XLRP) are one of the most common causes of severe visionloss. More than 25 years ago, the genetic loci were identified anddiscovery of the underlying gene defects followed. Human XLRP, caused bymutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene, is asevere early onset retinal degenerative disease that accounts for themajority of XLRP. Mutations in the retinitis pigmentosa GTPase regulator(RPGR) gene account for >70 % of the cases of XLRP, and exon ORF15, amutational hot spot in RPGR, is mutated in 22-60% of patients. Thedisease is relentlessly progressive, and by the end of their fourthdecade most patients are legally blind.

Until recently, progress has been slow in unraveling the molecularmechanisms that lead from mutation to PR degeneration, and in developingeffective treatments for most forms of RP, including RPGR-XLRP.Disease-relevant animal models have been crucial in developing andvalidating new therapies. For RPGR-XLRP there are both mouse and caninemodels. In the dog, two naturally-occurring distinct microdeletions inORF15 result in different disease phenotypes. X-linked progressiveretinal atrophy 1 (XLPRA1; del 1028-1032) has a C-terminus truncation of230 residues; the disease is juvenile, but post-developmental in onset,and progresses over several years. In contrast, the two-nucleotidedeletion associated with XLPRA2 (del 1084-1085) causes a frameshift, andinclusion of 34 basic amino acids that changes the isoelectric point ofthe putative protein, and truncates the terminal 161 residues. Thedisease is early onset and rapidly progressive. Both models correspondto the disease spectrum of human XLRP, and, although differing inrelative severity, they would be equivalent to human disease occurringwithin the first decade of life.

No successful treatment for XLRP is currently available to humanpatients suffering from this disease. What is needed is a treatment forRPGR-XLRP that is effective, safe and has long-term stability.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of preventing, arrestingprogression of or ameliorating vision loss associated with retinitispigmentosa in a subject. The method includes administering to thesubject an effective concentration of a composition comprising arecombinant adeno-associated virus (rAAV) carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which express the product of the gene inthe ocular cells of the subject, and a pharmaceutically acceptablecarrier.

In another aspect, a method of preventing or arresting progression ofphotoreceptor function loss, or increasing photoreceptor function in asubject in need thereof is provided. The method includes administeringto the subject an effective concentration of a composition comprising arAAV carrying a nucleic acid sequence encoding a normal RPGR gene, orfragment thereof, under the control of regulatory sequences whichexpress the product of the gene in the subject's ocular cells, and apharmaceutically acceptable carrier. As a result, the subject's visionis improved, or vision loss is arrested and/or ameliorated.

In another aspect, the invention provides a method of improvingphotoreceptor structure in a subject in need thereof. The methodincludes administering to the subject an effective concentration of acomposition comprising a rAAV carrying a nucleic acid sequence encodinga normal RPGR gene, or fragment thereof, under the control of regulatorysequences which express the product of the gene in the ocular cells ofthe subject, and a pharmaceutically acceptable carrier. As a result, thesubject's vision is improved, or vision loss is arrested and/orameliorated.

In yet another aspect, a method of preventing, arresting progression ofor ameliorating outer plexiform layer (OPL) abnormalities in a subjectin need thereof is provided. The method includes administering to thesubject an effective concentration of a composition comprising a rAAVcarrying a nucleic acid sequence encoding a normal RPGR gene, orfragment thereof, under the control of regulatory sequences whichexpress the product of the gene in the ocular cells of the subject, anda pharmaceutically acceptable carrier. As a result, the subject's visionis improved, or vision loss is arrested and/or ameliorated.

In another aspect, the invention provides a method of preventing,arresting progression of or ameliorating bipolar cell dendriteretraction in a subject in need thereof The method includesadministering to the subject an effective concentration of a compositioncomprising a rAAV carrying a nucleic acid sequence encoding a normalRPGR gene, or fragment thereof, under the control of regulatorysequences which express the product of the gene in the ocular cells ofthe subject, and a pharmaceutically acceptable carrier. As a result, thesubject's vision is improved, or vision loss is arrested and/orameliorated.

In another aspect, a method of preventing, arresting progression of orameliorating bipolar cell function loss in a subject in need thereof isprovided. The method includes administering to said subject an effectiveconcentration of a composition comprising a rAAV carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which express the product of the gene inthe ocular cells of the subject, and a pharmaceutically acceptablecarrier. As a result, the subject's vision is improved, or vision lossis arrested and/or ameliorated.

In yet another embodiment, the invention provides a method ofpreventing, arresting progression of or ameliorating axonal injurycharacterized by overexpression of neurofilaments in a subject in needthereof. The method includes administering to the subject an effectiveconcentration of a composition comprising a rAAV carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof under thecontrol of regulatory sequences which express the product of the gene inthe ocular cells of the subject, and a pharmaceutically acceptablecarrier. As a result, the subject's vision is improved, or vision lossis arrested and/or ameliorated.

In another aspect, a method of preventing XLRP in a subject at risk ofdeveloping the disease is provided. The method includes administering tothe subject an effective concentration of a composition comprising arAAV carrying a nucleic acid sequence encoding a normal RPGR gene, orfragment thereof, under the control of regulatory sequences whichexpress the product of the gene in the ocular cells of the subject, anda pharmaceutically acceptable carrier.

In yet another aspect, the invention provides a method of preventing,arresting or ameliorating rod and/or R/G cone opsin mislocalization in asubject in need thereof. The method includes administering to thesubject an effective concentration of a composition comprising a rAAVcarrying a nucleic acid sequence encoding a normal RPGR gene, orfragment thereof, under the control of regulatory sequences whichexpress the product of the gene in the ocular cells of the subject, anda pharmaceutically acceptable carrier. As a result, the subject's visionis improved, or vision loss is arrested and/or ameliorated.

In another aspect, the invention provides a method of preventing,arresting progression of or ameliorating OPL synaptic changes, bipolarcell abnormalities or inner retinal abnormalities associated withX-linked retinitis pigmentosa (XLRP) in a subject in need thereof. Themethod includes administering to the subject an effective concentrationof a composition comprising a rAAV carrying a nucleic acid sequenceencoding a normal RPGR gene, or fragment thereof, under the control ofregulatory sequences which express the product of the gene in thesubject's ocular cells, and a pharmaceutically acceptable carrier. As aresult, the subject's vision is improved, or vision loss is arrestedand/or ameliorated.

In yet another aspect, the invention provides a method of increasing orpreserving ONL thickness associated with XLRP in a subject in needthereof. The method includes administering to said subject an effectiveconcentration of a composition comprising a rAAV carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which express the product of the gene inthe ocular cells of the subject, and a pharmaceutically acceptablecarrier. As a result, the subject's vision is improved, or vision lossis arrested and/or ameliorated.

In another aspect, a method of treating or preventing XLRP in a subjectin need thereof is provided. The method includes (a) identifying subjecthaving, or at risk of developing, XLRP; (b) performing genotypicanalysis and identifying a mutation in the RPGR gene; (c) performingnon-invasive retinal imaging and functional studies and identifyingareas of retained photoreceptors that could be targeted for therapy; and(d) administering to the subject an effective concentration of acomposition comprising a recombinant virus carrying a nucleic acidsequence encoding a normal photoreceptor cell-specific gene under thecontrol of a promoter sequence which expresses the product of the genein the ocular cells of the subject, and a pharmaceutically acceptablecarrier, thereby preventing, arresting progression of or amelioratingXLRP.

In another aspect, the invention provides a composition comprising arecombinant adeno-associated virus (rAAV) carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which express the product of the gene inthe ocular cells of the subject, and a pharmaceutically acceptablecarrier.

In another aspect, the invention provides a composition including arecombinant AAV2/5 psendotyped adeno-associated virus, carrying anucleic acid sequence encoding a normal RPGR gene, or fragment thereof,under the control of a human IRBP or human GRK1 promoter which directsexpression of the product of the gene in the ocular cells of thesubject, formulated with a carrier and additional components suitablefor injection. In another embodiment, any of the methods described aboveutilizes this composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plasmid map of the AAV2/5 vector incorporating the hIRPBpromoter (SEQ ID NO: 2) and hRPGR1-ORF15 gene. The IRBP promoter in thisplasmid is a 241 bp fragment of the proximal promoter region of thehuman IRBP gene.

FIG. 2 is a plasmid map of the AAV2/5 vector incorporating the hGRK1promoter (SEQ ID NO: 3) and hRPGR1-ORF15 gene.

FIG. 3 is an alignment of the nucleic acid sequence encoding human RPGRvariant SEQ ID NO: 1 (bottom) and the nucleic acid sequence encodingvariant BK005711 (Wright) SEQ ID NO: 4 (top).

FIG. 4 is an alignment of the amino acid sequence of human RPGR variantSEQ ID NO: 5 (top) and the amino acid sequence of variant BK005711(Wright) SEQ ID NO: 6 (bottom).

FIG. 5 shows an alignment of amino acid residues aa 845-1039 of hRPGRvariant of SEQ ID NO: 5, with amino acids residues 845 -1052 of thevariant BK005711 of SEQ ID NO: 5, and further with the common aminoacids forming a consensus sequence SEQ ID NO: 7. The consensus sequencein the alignment appears with gaps at the positions of non-consensusamino acids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to various compositions and treatmentmethods utilizing the same comprising an effective concentration of arecombinant adeno-associated virus (rAAV) carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which direct expression of the productof the gene in the subject's ocular cells, formulated with a carrier andadditional components suitable for injection. The treatment methods aredirected to ocular disorders and associated conditions related thereto.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs and by reference to publishedtexts, which provide one skilled in the art with a general guide to manyof the terms used in the present application. The following definitionsare provided for clarity only and are not intended to limit the claimedinvention.

The terms “a” or “an” refers to one or more, for example, “a gene” isunderstood to represent one or more such genes. As such, the terms “a”(or “an”), “one or more,” and “at least one” are used interchangeablyherein. As used herein, the term “about” means a variability of 10% fromthe reference given, unless otherwise specified. With regard to thefollowing description, it is intended that each of the compositionsherein described, is useful, in another embodiment, in the methods ofthe invention. In addition, it is also intended that each of thecompositions herein described as useful in the methods, is, in anotherembodiment, itself an embodiment of the invention. While variousembodiments in the specification are presented using “comprising”language, under other circumstances, a related embodiment is alsointended to be interpreted and described using “consisting of” or“consisting essentially of” language.

A. THE MAMMALIAN SUBJECT

As used herein, the term “mammalian subject” or “subject” includes anymammal in need of these methods of treatment or prophylaxis, includingparticularly humans. Other mammals in need of such treatment orprophylaxis include dogs, cats, or other domesticated animals, horses,livestock, laboratory animals, including non-human primates, etc. Thesubject may be male or female. In one embodiment, the subject has, or isat risk of developing, RP and more particularly, XLRP. In anotherembodiment, the subject is a “carrier” for XLRP, has at least one RPGRmutation in at least one X chromosome. Because XLRP is an X-linkeddisease, females, which normally have two X chromosomes, may behomozygous or heterozygous for a specific mutation in the RPGR gene, orcompound heterozygotes, which have a different mutation in the RPGR geneon each X chromosome. Normal males, having only one X chromosome, with amutation in the RPGR gene are termed hemizygous. In one embodiment, thesubject having, or at risk of developing XLRP is a hemizygous male. Inanother embodiment, the subject having, or at risk of developing XLRP,is a homozygous female or a heterozygous female. In other embodiments,subjects at risk of developing XLRP include those with a family historyof XLRP, those with one or more confirmed mutations in the RPGR gene,offspring of female carriers of an RPGR mutation (heterozygous females),or offspring of females carrying an RPGR mutation on both X chromosomes.

In another embodiment, the subject has shown clinical signs of XLRP.Clinical signs of XLRP include, but are not limited, to, decreasedperipheral vision, decreased central (reading) vision, decreased nightvision, loss of color perception, reduction in visual acuity, decreasedphotoreceptor function, pigmentary changes. In another embodiment, thesubject has been diagnosed with XLRP. In yet another embodiment, thesubject has not yet shown clinical signs of XLRP.

In yet another embodiment, the subject has 10% or more photoreceptordamage/loss. In another embodiment, the subject has 20% or morephotoreceptor damage/loss. In another embodiment, the subject has 30% ormore photoreceptor damage/loss. In another embodiment, the subject has40% or more photoreceptor damage/loss. In another embodiment, thesubject has 50% or more photoreceptor damage/loss. In anotherembodiment, the subject has 60% or more photoreceptor damage/loss. Inanother embodiment, the subject has 70% or more photoreceptordamage/loss. In another embodiment, the subject has 80% or morephotoreceptor damage/loss. In another embodiment, the subject has 90% ormore photoreceptor damage/loss.

B. XLRP AND RPGR

In one aspect, the methods herein relate to the treatment or preventionof retinitis pigmentosa (RP). In an embodiment, the retinitis pigmentosais an X-linked retinitis pigmentosa (XLRP). XLRP is one of the mostsevere forms of RP, demonstrating an early age of onset (usually withinthe first decade) and rapid progression of disease. Because the diseaseis X-linked, homozygous females are rare, usually only manifesting insmall, isolated populations. Thus, the disease primarily affects males,although carrier (heterozygous) females are also affected, demonstratingvarious levels of retinal degeneration. The disease demonstrates a broadspectrum of disease severity, between and within families.

The retinitis pigmentosa GTPase regulator (RPGR) is an 815 aa proteinwhich is implicated in XLRP (Meindl et al, Nature Genetics, 13:35-42(May 1996) and Vervoort et al, Nature Genetics, 25:462-6 (2000), whichare hereby incorporated by reference herein). Greater than 290 mutationsin RPGR (http://rpgr.hgu.mrc.ac.uk/supplementary/, and document entitled“Summary of RPGR mutation and polymorphism”, which is incorporatedherein by reference) account for over 70% of XERP patients. The proteincontains a RCC-1 like domain, characteristic of the highly conservedguanine nucleotide exchange factors. The constitutive transcript ofRPGR, containing 19 exons, is expressed in a wide variety of tissues(Hong and Li, Invest Ophthalmology Vis Sci, 43(11):3373-82, incorporatedby reference herein). An RPGR variant terminates in intron 15 of theRPGR gene. The alternative terminal exon consists of the constitutiveexon 15 and part of intron 15, and is termed ORF15. This protein isoformthat is encoded by exons 1 through ORF15 is used prevalently inphotoreceptors and a large number of disease causing mutations have beenfound in ORF15 (Vervoort and Wright, Hum Mutat. 2002 May, 19(5):486-500;Aguirre et al, Exp Eye Res, 2002, 75:431-43; and Neidhardt et al, HumMutat. 2007, 28(8):797-807, each of which is hereby incorporated byreference herein).

In one aspect the method employs a nucleic acid sequence encoding anormal RPGR gene, or fragment thereof. The term “RPGR” as used herein,refers to the full length gene itself or a functional fragment, asfarther defined below. The nucleic acid sequence encoding a normal RPGRgene may be derived from any mammal which natively expresses the RPGRgene, or homolog thereof. In another embodiment, the RPGR gene sequenceis derived from the same mammal that the composition is intended totreat. In another embodiment, the RPGR is derived from a human. Inanother embodiment, the RPGR sequence is the sequence of the full lengthhuman RPGRORF15 clone, which includes exons 1 though ORF15 (Vervoort R,et al. (2000), Nat Genet 25:462-466, which is incorporated by referenceherein). In another embodiment, the RPGR sequence is that shown in SEQID NO: 1. In another embodiment, the RPGR sequence is a functionalfragment of the RPGRORF15 clone. By the term “fragment” or “functionalfragment”, it is meant any fragment that retains the function of thefull length clone, although not necessarily at the same level ofexpression or activity. For example, acDNA representing RPGR-ORF15 butshortened by 654 bp in the repetitive region has been shown toreconstitute RPGR function in mice. (Hong, D. H. et al, InvestOphthalmol Vis Sci 46(2): 435-441 which is hereby incorporated, byreference). Similar functional fragments of human, or other RPGRsequences may be determined by one of skill in the art. In anotherembodiment, the RPGR is derived from a canine. In other embodiments,certain modifications are made to the RPGR sequence in order to enhancethe expression in the target cell. Such modifications include codonoptimization, (see, e.g., U.S. Pat. Nos. 7,561,972; 7,561,973; and7,888,112, incorporated herein by reference) and conversion of thesequence surrounding the translational start site to a consensus Kozaksequence: geeReeATGR. See, Kozak et al, Nucleic Acids Res. 15 (20):8125-8148, incorporated herein by reference.

As used herein, the term “ocular cells” refers to any cell in, orassociated with the function of, the eye. The term may refer to any oneof photoreceptor cells, including rod, cone and photosensitive ganglioncells or retinal pigment epithelium (RPE) cells. In one embodiment, theocular cells are the photoreceptor cells.

C. AAV VECTORS AND COMPOSITIONS

In certain embodiments of this invention, the RPGR nucleic acidsequence, or fragment thereof, is delivered to the ocular cells in needof treatment by means of a viral vector, of which many are known andavailable in the art. For delivery to the ocular cells, the therapeuticvector is desirably non-toxic, non-immunogenic, easy to produce, andefficient in protecting and delivering DNA into the target cells. In oneparticular embodiment, the viral vector is an adeno-associated virusvector. In another embodiment, the invention provides a therapeuticcomposition comprising an adeno-associated viral vector comprising anRPGR sequence under the control of a suitable promoter. In oneembodiment, the RPGR sequence is encoded by SEQ ID NO: 1.

More than 30 naturally occurring serotypes of AAV are available. Manynatural variants in the AAV capsid exist, allowing identification anduse of an AAV with properties specifically suited for ocular cells. AAVviruses may be engineered by conventional molecular biology techniques,making it possible to optimize these particles for cell specificdelivery of RPGR nucleic acid sequences, for minimizing immunogenicity,for tuning stability and particle lifetime, for efficient degradation,for accurate delivery to the nucleus, etc.

Thus, RPGR overexpression can be achieved in the ocular cells throughdelivery by recombinantly engineered AAVs or artificial AAV's thatcontain sequences encoding RPGR. The use of AAVs is a common mode ofexoamous delivery of DNA as it is relatively non-toxic, providesefficient gene transfer, and can be easily optimized for specificpurposes. Among the serotypes AAVs isolated from human or non-humanprimates (NHP) and well characterized, human serotype 2 is the first AAVthat was developed as a gene transfer vector; it has been widely usedfor efficient gene transfer experiments in different target tissues andanimal models. Clinical trials of the experimental application of AAV2based vectors to some human disease models are in progress, and includesuch diseases as cystic fibrosis and hemophilia B. Other AAV serotypesinclude, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8 and AAV9. See, e.g., WO 2005/033321 for a discussion of various AAVserotypes, which is incorporated herein by reference.

Desirable AAV fragments for assembly into vectors include the capproteins, including the vp1, vp2, vp3 and hypervariable regions, the repproteins, including rep 78, rep 68, rep 52, and rep 40, and thesequences encoding these proteins. These fragments may be readilyutilized in a variety of vector systems and host cells. Such fragmentsmay be used alone, in combination with other AAV serotype sequences orfragments, or in combination with elements from other AAV non-AAV viralsequences. As used herein, artificial AAV serotypes include, withoutlimitation, AAV with anon-naturally occurring capsid protein. Such anartificial capsid may be generated by any suitable technique, using aselected AAV sequence (e.g., a fragment of a vp1 capsid protein) incombination with heterologous sequences which may be obtained from adifferent selected AAV serotype, non-contiguous portions of the same AAVserotype, from a non-AAV viral source, or from anon-viral source. Anartificial AAV serotype may be, without limitation, a pseudotyped AAV, achimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAVcapsid. Pseudotyped vectors, wherein the capsid of one AAV is replacedwith a heterologous capsid protein, are useful in the invention. Forillustrative purposes, AAV2/5 is used in the examples described below.In a preferred embodiment, the AAV is AAV2/5. In another preferredembodiment, the AAV is AAV2/8. See, Mussolino et al, cited above.

In one embodiment, the vectors useful in compositions and methodsdescribed herein contain, at a minimum, sequences encoding a selectedAAV serotype capsid, e.g., an AAV5 capsid, or a fragment thereof. Inanother embodiment, useful vectors contain, at a minimum, sequencesencoding a selected AAV serotype rep protein, e.g., AAV5 rep protein, ora fragment thereof. Optionally, such vectors may contain both AAV capand rep proteins. In vectors in which both AAV rep and cap are provided,the AAV rep and AAV cap sequences can both be of one serotype e.g., allAAV5 origin. Alternatively, vectors may be used in which the repsequences are from an AAV serotype which differs from that which isproviding the cap sequences. In one embodiment, the rep and capsequences are expressed from separate sources (e.g., separate vectors,or a host cell and a vector). In another embodiment, these rep sequencesare fused in frame to cap sequences of a different AAV serotype to forma chimeric AAV vector, such as AAV2/8 described in U.S. Pat. No.7,282,199, which is incorporated by reference herein.

A suitable recombinant adeno-associated virus (AAV) is generated byculturing a host cell which contains a nucleic acid sequence encoding anadeno-associated virus (AAV) serotype capsid protein, or fragmentthereof, as defined herein; a functional rep gene; a minigene composedof, at a minimum, AAV inverted terminal repeats (ITRs) and a RPGRnucleic acid sequence; and sufficient helper functions to permitpackaging of the minigene into the AAV capsid protein. The componentsrequired to be cultured in the host cell to package an AAV minigene inan AAV capsid may be provided to the host cell in trans. Alternatively,any one or more of the required components (e.g., minigene, repsequences, cap sequences, and/or helper functions) may be provided by astable host cell which has been engineered to contain one or more of therequired components using methods known to those of skill in the art.

Most suitably, such a stable host cell will contain the requiredcomponent(s) under the control of an inducible promoter. However, therequired component(s) may be under the control of a constitutivepromoter. Examples of suitable inducible and constitutive promoters areprovided herein, in the discussion below of regulatory elements suitablefor use with the transgene, i.e., RPGR. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated which is derived from 293 cells (whichcontain E1 helper functions under the control of a constitutivepromoter), but which contains the rep and/or cap proteins under thecontrol of inducible promoters. Still other stable host cells may begenerated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transfersthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, 1993 J. Viral., 70:520-532 and U.S. Pat. No. 5,478,745,among others. These publications are incorporated by reference herein.

Unless otherwise specified, the AAV ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAVserotype, including, without limitation, AAV1, AAV2, AAV3, AAV5, AAV6,AAV7, AAV8, AAV9 or other known and unknown AAV serotypes. These ITRs orother AAV components may be readily isolated using techniques availableto those of skill in the art from an AAV serotype. Such AAV may beisolated or obtained from academic, commercial, or public sources (e.g.,the American Type Culture Collection, Manassas, Va.). Alternatively, theAAV sequences may be obtained through synthetic or other suitable meansby reference to published sequences such as are available in theliterature or in databases such as, e.g., GenBank, PubMed, or the like.

The minigene is composed of, at a minimum, RPGR nucleic acid sequence(the transgene), as described above, and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). In one desirableembodiment, the ITRs of AAV serotype 2 are used. However, ITRs fromother suitable serotypes may be selected. It is this minigene which ispackaged into a capsid protein and delivered to a selected host cell.

The regulatory sequences include conventional control elements which areoperably linked to the RPGR acne in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe vector or infected with the virus produced by the invention. As usedherein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters, are known in the art and may beutilized.

The regulatory sequences useful in the constructs of the presentinvention may also contain an in tron, desirably located between thepromoter/enhancer sequence and the gene. One desirable intron sequenceis derived from SV-40, and is a 100 bp mini-intron splice donor/spliceacceptor referred to as SD-SA. Another suitable sequence includes thewoodchuck hepatitis virus post-transcriptional element. (See, e.g., L.Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910).PolyA signals may be derived from many suitable species, including,without limitation SV-40, human and bovine.

Another regulatory component of the rAAV useful in the method of theinvention is an internal ribosome entry site (IRES). An IRES sequence,or other suitable systems, may be used to produce more than onepolypeptide from a single gene transcript. An IRES (or other suitablesequence) is used to produce a protein that contains more than onepolypeptide chain or to express two different proteins from or withinthe same cell. An exemplary IRES is the poliovirus internal ribosomeentry sequence, which supports transgene expression in photoreceptors,RPE and ganglion cells. Preferably, the IRES is located 3′ to thetransgene in the rAAV vector.

The selection of the promoter to be employed in the rAAV may be madefrom among a wide number of constitutive or inducible promoters that canexpress the selected transgene in the desired an ocular cell. In anotherembodiment, the promoter is cell-specific. The term “cell-specific”means that the particular promoter selected for the recombinant vectorcan direct expression of the selected transgene in a particular ocularcell type. In one embodiment, the promoter is specific for expression ofthe transgene in photoreceptor cells. In another embodiment, thepromoter is specific for expression in the rods and cones. In anotherembodiment, the promoter is specific for expression in the rods. Inanother embodiment, the promoter is specific for expression in thecones. In another embodiment, the promoter is specific for expression ofthe transgene RPE cells. In another embodiment, the transgene isexpressed in any of the above noted ocular cells.

The promoter may be derived from any species. In another embodiment, thepromoter is the human G-protein-coupled receptor protein kinase 1 (GRK1)promoter (Genbank Accession number AY327580). In another embodiment, thepromoter is a 292 nt fragment (positions 1793-2087) of the GRK1 promoter(SEQ NO: 2) (See also, Beltran et al, Gene Therapy 2010 17:1162-74,which is hereby incorporated by reference herein). In another preferredembodiment, the promoter is the human interphotoreceptorretinoid-binding protein proximal (IRBP) promoter. In one embodiment,the promoter is a 235 nt fragment of the hIRBP promoter (SEQ NO: 3). Inanother embodiment, promoter is the native promoter for the gene to beexpressed. In one embodiment, the promoter is the RPGR proximal promoter(Shu et al, IOVS, May 2102, which is incorporated by reference herein).Other promoters useful in the invention include, without limitation, therod opsin promoter, the red-green opsin promoter, the blue opsinpromoter, the cGMP-β-phosphodiesterase promoter, the mouse opsinpromoter (Beltran et al 2010 cited above), the rhodopsin promoter(Mussolino et al, Gene Ther, July 2011, 18(7):637-45); the alpha-subunitof cone transducin (Morrissey et al, BMC Dev, Biol, January 2011, 11:3);beta phosphodiesterase (PDE) promoter; the retinitis pimentosa (RP1)promoter (Nicord et al, J. Gene Med, December 2007, 9(12):1015-23); theNXNL2,/NXNL1 promoter (Lombard et al, PLoS One, October 2010,5(10):e13025), the RPE65 promoter; the retinal degenerationslow/peripherin 2 (Rds/perph2) promoter (Cai et al, Exp Eye Res. 2010August; 91(2):186-94); and the VMD2 promoter (Kochi et al, Human GeneTherapy, 2009(20:31-9)). Each of these documents is incorporated byreference herein. In one embodiment, the promoter is of a small size,under 1000 bp, due to the size limitations of the AAV vector. In anotherembodiment, the promoter is under 400 bp.

Examples of constitutive promoters useful in the invention include,without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter(optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter(optionally with the CMV enhancer), the SV40 promoter, the dihydrofolatereductase promoter, the chicken β-actin (CBA) promoter, thephosphoglycerol kinase (PGK) promoter, the EF 1promoter (Invitrogen),and the immediate early CMV enhancer coupled with the CBA promoter(Beltran et al, Gene Therapy 2010 cited above).

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, acutephase, a particular differentiation state of the cell, or in replicatingcells only. Inducible promoters and inducible systems are available froma variety of commercial sources, including, without limitation,Invitrogen, Clontech and Ariad. Many other systems have been describedand can be readily selected by one of skill in the art. Examples ofinducible promoters regulated by exogenously supplied compounds,include, the zinc-inducible sheep metallothionine (MT) promoter, thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,the T7 polymerase promoter system; the ecdysone insect promoter, thetetracycline-repressible system, the tetracycline-inducible system, theRU486-inducible system and the rapamycin-inducible system. Other typesof inducible promoters which may be useful in this context are thosewhich are regulated by a specific physiological state, e.g.,temperature, acute phase, a particular differentiation state of thecell, or in replicating cells only. Any type of inducible promoter whichis tightly regulated and is specific for the particular target ocularcell type may be used.

Other regulatory sequences useful in the invention include enhancersequences. Enhancer sequences useful in the invention include the IRBPenhancer (Nicord 2007, cited above), immediate early cytomegalovirusenhancer, one derived from an immunoglobulin gene or SV40 enhancer, thecis-acting element identified in the mouse proximal promoter, etc.

Selection of these and other common vector and regulatory elements areconventional and many such sequences are available. See, e.g., Sambrooket al, and references cited therein at, for example, pages 3.18-3.26 and16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1989). Of course, not all vectors andexpression control sequences will function equally well to express allof the transgenes of this invention. However, one of skill in the artmay make a selection among these, and other, expression controlsequences without departing from the scope of this invention.

D. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

The recombinant AAV containing the desired transgene and cell-specificpromoter for use in the target ocular cells as detailed above ispreferably assessed for contamination by conventional methods and thenformulated into a pharmaceutical composition intended for subretinalinjection. Such formulation involves the use of a pharmaceuticallyand/or physiologically acceptable vehicle or carrier, particularly onesuitable for administration to the eye, e.g., by subretinal injection,such as buffered saline or other buffers, e.g., HEPES, to maintain pH atappropriate physiological levels, and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. Exemplary physiologically acceptable carriers include sterile,pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.A variety of such known carriers are provided in U.S. Pat. PublicationNo. 7,629,322, incorporated herein by reference. In one embodiment, thecarrier is an isotonic sodium chloride solution. In another embodiment,the carrier is balanced salt solution. In one embodiment, the carrierincludes tween. If the virus is to be stored long-term, it may be frozenin the presence of glycerol or Tween20.

In certain embodiments of the methods of this invention, thepharmaceutical composition described above is administered to thesubject by subretinal injection. The use of subretinal injection as theroute of delivery is a critical component of this method, asintravitreal administration currently does not enable the sametherapeutic effects.

Furthermore, in certain embodiments of the invention it is desirable toperform non-invasive retinal imaging and functional studies to identifyareas of retained photoreceptors to be targeted for therapy. In theseembodiments, clinical diagnostic tests are employed to determine theprecise location(s) for one or more subretinal injection(s). These testsmay include electroretinography (ERG), perimetry, topographical mappingof the layers of the retina and measurement of the thickness of itslayers by means of confocal scanning laser ophthalmoscopy (cSLO) andoptical coherence tomography (OCT), topographical mapping of conedensity via adaptive optics (AO), functional eye exam, etc. These, andother desirable tests, are described in the examples below. In view ofthe imaging and functional studies, in some embodiments of the inventionone or more injections are performed in the same eye in order to targetdifferent areas of retained photoreceptors. The volume and viral titerof each injection is determined individually, as further describedbelow, and may be the same or different from other injections performedin the same, or contralateral, eye. In another embodiment, a single,larger volume injection is made in order to treat the entire eye. In oneembodiment, the volume and concentration of the rAAV composition isselected so that only the region of damaged photoreceptors is impacted.In another embodiment, the volume and/or concentration of the rAAVcomposition is a greater amount, in order reach larger portions of theeye, including non-damaged photoreceptors.

The composition may be delivered in a volume of from about 50 μL toabout 1 mL, including all numbers within the range, depending on thesize of the area to be treated, the viral titer used, the route ofadministration, and the desired effect of the method. In one embodiment,the volume is about 50 μL. In another embodiment, the volume is about 70μL. In another embodiment, the volume is about 100 μL. In anotherembodiment, the volume is about 125 μL. In another embodiment, thevolume is about 150 μL. In another embodiment, the volume is about 175μL. In yet another embodiment, the volume is about 200 μL. In anotherembodiment, the volume is about 250 μL. In another embodiment, thevolume is about 300 μL. In another embodiment, the volume is about 450μL. In another embodiment, the volume is about 500 μL. In anotherembodiment, the volume is about 600 μL. In another embodiment, thevolume is about 750 μL. In another embodiment, the volume is about 850μL. In another embodiment, the volume is about 1000 μL. An effectiveconcentration of a recombinant adeno-associated virus carrying a nucleicacid sequence encoding the desired transgene under the control of thecell-specific promoter sequence desirably ranges between about 10⁸ and10¹³ vector genomes per milliliter (vg/mL). The rAAV infectious unitsare measured as described in S. K. McLaughlin et al, 1988 J. Virol.,62:1963. Preferably, the concentration is from about 1.5×10⁹ vg/mL toabout 1.5×10¹² vg/mL, and more preferably from about 1.5×10⁹ vg/mL toabout 1.5×10¹¹ vg/mL. In one embodiment, the effective concentration isabout 1.5×10¹⁰ vg/mL. In another embodiment, the effective concentrationis about 1.5×10¹¹ vg/mL. In another embodiment, the effectiveconcentration is about 2.8×10¹¹ vg/mL. In yet another embodiment, theeffective concentration is about 1.5×10¹² vg/mL. In another embodiment,the effective concentration is about 1.5×10¹³ vg/mL. It is desirablethat the lowest effective concentration of virus be utilized in order toreduce the risk of undesirable effects, such as toxicity, retinaldysplasia and detachment. Still other dosages in these ranges may beselected by the attending physician, taking into account the physicalstate of the subject, preferably human, being treated, the age of thesubject, the particular ocular disorder and the degree to which thedisorder, if progressive, has developed.

E. METHODS OF TREATMENT/PROPHYLAXIS

The invention provides various methods of preventing, treating,arresting progression of or ameliorating the above-described oculardiseases and retinal changes associated therewith. Generally, themethods include administering to a mammalian subject in need thereof, aneffective amount of a composition comprising a recombinantadeno-associated virus (AAV) carrying a nucleic acid sequence encoding anormal RPGR gene, or fragment thereof, under the control of regulatorysequences which express the product of the gene in the subject's ocularcells, and a pharmaceutically acceptable carrier.

RP, and more particularly, XLRP is associated with many retinal changes.These include a loss of photoreceptor structure or function; thinning orthickening of the outer nuclear layer (ONL); thinning or thickening ofthe outer plexiform layer (OPL); disorganization followed by loss of rodand cone outer segments; shortening of the rod and cone inner segments;retraction of bipolar cell dendrites; thinning or thickening of theinner retinal layers including inner nuclear layer, inner plexiformlayer, ganglion cell layer and nerve fiber layer; opsin mislocalization;overexpression of neurofilaments; loss of ERG function; loss of visualacuity and contrast sensitivity; and loss of visually guided behavior.In one embodiment, the invention provides a method of preventing,arresting progression of or ameliorating any of the retinal changesassociated with XLRP. As a result, the subject's vision is improved, orvision loss is arrested and/or ameliorated.

In a particular embodiment, the invention provides a method ofpreventing, arresting progression of or ameliorating vision lossassociated with retinitis pigmentosa in the subject. Vision lossassociated with retinitis pigmentosa refers to any decrease inperipheral vision, central (reading) vision, night vision, day vision,loss of color perception, loss of contrast sensitivity, or reduction invisual acuity.

In another embodiment, the invention provides a method to prevent, orarrest photoreceptor function loss, or increase photoreceptor functionin the subject. Photoreceptor function may be assessed using thefunctional studies described above and in the examples below, e.g., ERGor perimetry, which are conventional in the art. As used herein“photoreceptor function loss” means a decrease in photoreceptor functionas compared to a normal, non-diseased eye or the same eye at an earliertime point As used herein, “increase photoreceptor function” means toimprove the function of the photoreceptors or increase the number orpercentage of functional photoreceptors as compared to a diseased eye(having the same ocular disease), the same eye at an earlier time point,a non-treated portion of the same eye, or the contralateral eye of thesame patient.

In another aspect, the invention provides method of improvingphotoreceptor structure in the subject. As used herein “improvingphotoreceptor structure” refers (in the region of the retina that istreated) to one or more of an increase or decrease in outer nuclearlayer (ONL) thickness, or arresting progression of ONL thickening orthinning, across the entire retina, in the central retina, or theperiphery; increase or decrease in outer plexiform layer (OPL)thickness, or arresting progression of OPL thickening or thinning,across the entire retina, in the central retina, or the periphery;decrease in rod and cone inner segment (IS) shortening; decrease inshortening and loss of outer segments (OS); decrease in bipolar celldendrite retraction, or an increase in bipolar cell dendrite length oramount; and reversal of opsin mislocalization.

In another aspect, the invention provides a method of preventing,arresting progression of or ameliorating abnormalities of the outerplexiform layer (OPL) in a subject in need thereof. As used herein, toameliorate abnormalities of the OPL means (in the region of the retinathat is treated) to increase or decrease the OPL thickness, or arrest ofOPL thickness changes, across the entire retina, in the central retina,or the periphery. In another aspect, the invention provides a method ofincreasing, decreaseing or preserving ONL thickness associated withX-linked form of retinitis pigmentosa (XLRP) in a subject in needthereof. Progressive ONL thinning is common in all phenotypes of XLRP,and is sometimes proceeded by abnormal ONL thickening As used herein,“increasing, decreasing or preserving ONL thickness” means to increaseONL thickness if it is thinner than normal, decrease ONL thickness if itis thicker than normal, or arresting progression of ONL thinning, acrossthe entire retina, in the central retina, or the periphery. In anotheraspect, the invention provides a method of preventing, arrestingprogression of or ameliorating bipolar cell dendrite retraction in asubject in need thereof. In the course of XLRP progression, bipolar celldendrites retract and fail to connect with photoreceptor cells. Theinventors made the surprising discovery that, after treatment with therAAV-RPGR construct described above, bipolar cells in damaged areas formnew dendrites which are able to connect to the photoreceptor cells andimprove function. Thus, in another embodiment, the invention provides amethod of preventing, arresting progression of or ameliorating orimproving bipolar cell function loss in a subject in need thereof.Enhancement of biolar function also leads to improved scotopic (rodmediated.) ERG b-wave amplitudes. In another embodiment, the inventionprovides a method of improving post receptoral responses for rods andcones as recorded by ERG. In another aspect, the invention provides amethod of preventing, arresting progression of or ameliorating axonalinjury characterized by overexpression of neurofilaments in a subject inneed thereof.

In another aspect, the invention provides a method of preventingX-linked retinitis pigmentosa (XLRP) in a subject at risk of developingsaid disease. Subjects at risk of developing XLRP include those with afamily history of XLRP, those with one or more confirmed mutations inthe RPGR gene, offspring of female carriers of an RPGR mutation(heterozygous females), offspring of females carrying an RPGR mutationon both X chromosomes.

In another aspect, the invention provides a method of preventing,arresting progression of or ameliorating rod and/or R/G cone opsinmislocalization in a subject in need thereof. In normal eye, rhodopsinand R/G cone opsin are found predominantly in membranes of the rod cellouter segment but become mislocalized to the inner segment, ONL and/orthe synaptic terminals in many retinal diseases and injuries, includingXLRP.

In another aspect, the invention provides a method of preventing,arresting or ameliorating the increase in phagocytic cells in thesubretinal space at later stages of the disease. See, Beltran et al,IOVS (2006) 47:1669-81, incorporated herein by reference herein.

In another aspect, the invention provides a method of preventing,arresting progression of or ameliorating OPL synaptic changes, bipolarcell abnormalities or inner retinal abnormalities associated with XLRPin a subject in need thereof. OPL synaptic changes include narrowing ofthe OPL associated with compressed photoreceptor synaptic terminals, anda reduction of the number of CtBP2-labeled synaptic ribbons in rod andcone terminals. Bipolar cell abnormalities include retraction of bipolarcell dendrites. Inner retinal abnormalities include inner retinalhyperthickness, rod photoreceptor neurite sprouting, rod bipolar celldendrite retraction, increased GABA-immunoreactive amacrine cells, andchanges in Müller glial cell reactivity, flattening of the axonalarborization of horizontal cells that can be labeled with a calbindinantibody; thinning and loss of lamination (at later stages of disease)of the inner plexiform layer (IPL) that can be tabled with GABAantibody. See, Beltran et al, IOVS, 2006, cited above and Aleman et al,IOVS (2007) 48:4759-65 incorporated by reference herein.

For each of the described methods, the treatment may be used to preventthe occurrence of retinal damage or to rescue eyes having mild oradvanced disease. As used herein, the term “rescue” means to preventprogression of the disease to total blindness, prevent spread of damageto uninjured photoreceptor cells or to improve damage in injuredphotoreceptor cells. Thus, in one embodiment, the composition isadministered before disease onset. In another embodiment, thecomposition is administered after the initiation of opsinmislocalization. In another embodiment, the composition is administeredprior to the initiation of photoreceptor loss. In another embodiment,the composition is administered after initiation of photoreceptor loss.In yet another embodiment, the composition is administered when lessthan 90% of the photoreceptors are functioning or remaining, as comparedto a non-diseased eye. In another embodiment, the composition isadministered when less than 80% of the photoreceptors are functioning orremaining. In another embodiment, the composition is administered whenless than 70% of the photoreceptors are functioning or remaining. Inanother embodiment, the composition is administered when less than 60%of the photoreceptors are functioning or remaining. In anotherembodiment, the composition is administered when less than 50% of thephotoreceptors are functioning or remaining. In another embodiment, thecomposition is administered when less than 40% of the photoreceptors arefunctioning or remaining. In another embodiment, the composition isadministered when less than 30% of the photoreceptors are functioning orremaining. In another embodiment, the composition is administered whenless than 20% of the photoreceptors are functioning or remaining. Inanother embodiment, the composition is administered when less than 10%of the photoreceptors are functioning or remaining. In one embodiment,the composition is administered only to one or more regions of the eye,e.g., those which have retained photoreceptors. In another embodiment,the composition is administered to the entire eye.

In another embodiment, a method of treating or preventing XLRP in asubject in need thereof is provided. The method includes identifying asubject having, or at risk of developing, XLRP; performing genotypicanalysis and identifying at least one mutation in the RPGR gene;performing non-invasive retinal imaging and functional studies andidentifying areas of retained photoreceptors to be targeted for therapy;and administering to the subject an effective concentration of acomposition, whereby XLRP is prevented, arrested or ameliorated. Thecomposition includes a recombinant virus carrying a nucleic acidsequence encoding a normal photoreceptor cell-specific gene under thecontrol of a promoter sequence which expresses the product of the genein the photoreceptor cells, and a pharmaceutically acceptable carrier.Genotypic analysis is routine in the art and may include the use of PCRto identify one or more mutations in the nucleic acid sequence of theRPGR gene. See, e,g., Meindl et al, Nat Gen, May 1996, 13:35, Vervoort,R. et al, 2000. Nat Genet 25(4): 462-466 (cited above); and Vervoort, R.and Wright, A. F. 2002, Human Mutation 19: 486-500, each of which isincorporated herein by reference.

In another embodiment, any of the above methods are performed utilizinga composition comprising a recombinant AAV2/5 pseudotypedadeno-associated virus, carrying a nucleic acid sequence encoding anormal RPGR gene, or fragment thereof, under the control of an IRBP orGRK1 promoter which directs expression of the product of the gene in thephotoreceptor cells of the subject, formulated with a carrier andadditional components suitable for subretinal injection.

In another embodiment of the invention, the method includes performingfunctional and imaging studies to determine the efficacy of thetreatment. These studies include ERG and in vivo retinal imaging, asdescribed in the examples below. In addition visual field studies,perimetry, and microperimetry, mobility testing, visual acuity, colorvision testing may be performed.

In yet another embodiment of the invention, any of the above describedmethods is performed in combination with another, or secondary, therapy.The therapy may be any now known, or as yet unknown, therapy which helpsprevent, arrest or ameliorate XLRP or any of the above-described effectsassociated therewith. In one embodiment, the secondary therapy isCiliary Neurotrophic Factor (CNTF). Sieving, P. A. et al, 2006. ProcNatl Acad Sci USA 103(10): 3896-3901, which is hereby incorporated byreference. The secondary therapy can be administered before, concurrentwith, or after administration of the rAAV described above.

As is demonstrated in the examples below, an exemplary hRPGRORF15 wasemployed in in vivo experiments to provide evidence of the utility andefficacy of the methods and compositions of this invention. The examplesdemonstrated restoration of retinal function by the method of thisinvention in a large animal model of a human retinopathy. The use of theexemplary vector demonstrated in the experiments that the defect in theRPGR mutant dogs could be corrected by gene delivery. Retinal functionwas improved in this large animal model of blindness. This data allowone of skill in the art to readily anticipate that this method may besimilarly used in treatment of XLRP or other types of retinal disease inother subjects, including humans.

F. EXAMPLES Example 1 Materials and Methods

a. Human Subjects and Retinal Cross-Sectional Imaging

Patients with XLRP and molecularly confirmed RPGRORF15 mutations wereincluded in this study. Informed consent was obtained. Proceduresfollowed the Declaration of Helsinki guidelines and were approved by theinstitutional review board. Retinal cross-sectional imaging was obtainedwith spectral-domain optical coherence tomography (SD-OCT, RTVue-100;Optovue Inc., Fremont, Calif.). Recording and analysis techniques werepublished previously.

b. Animals

To define the structural and functional consequences of XLPRA1 andXLPRA2 disease and set the stage for treatment and outcome assessment,we used wild type (n-17, ages 7-416 wks), XLPRA1 (n=9, ages 7-156 wks)and XLPRA2 (n=6, ages 8-144 wks) dogs for non-invasive imaging and ERGstudies. For gene therapy, crossbred affected dogs were used (Tables 1 &2 below). All procedures involving animals were performed in compliancewith the ARVO Statement for the Use of Animals in Ophthalmic and VisionResearch and IACUC approval.

c. rAAV Vector Production and Purification

A full length human RPGRORF15 was cloned into AAV2/5 viral vectors andregulated by the human IRBP or GRK1 promoters. The vector cDNA was afull length human RPGRORF15 clone, based on the sequence published byAlan Wright and colleagues (BK005711)(http://www.ncbi.nlm.nih.gov/nuccore/BK005711), incorporated byreference herein. However, the sequence used in the construct (SEQ IDNO: 1) exhibits greater stability than the Wright sequence. Thisconstruct contains exons 1-ORF15, and was generated using 3-way ligationby step-wise amplifying exons 1-part of 15b (nucleotides 169-1990) fromhuman lymphocytes and 1991-3627 from human genomic DNA. An internalrestriction enzyme site Nde1 (CATATG) was created by site-directedmutagenesis at residue 1993 (A>T). The RPGRORF15 sequence used in theconstructs is shown in SEQ ID NO: 1. An alignment of the nucleic acidsequence published by Alan Wright and colleagues (BK005711) and SEQ IDNO: 1 is provided in FIG. 3 (See, also the aligned amino acid sequencesencoded by the two nucleic acid sequences in FIG. 4, and a partialconsensus sequence illustrated in FIG. 5). These fragments were thencloned in BamHI and XhoI sites in pBluescript, propagated in E. coliStb14 and sequence-verified at the University of Michigan DNA sequencingcore facility.

The human G-protein-coupled receptor protein kinase 1 (hGRK1) promoterwas used to primarily control rod expression in the dog retina at atherapeutic concentration of 10¹¹ vg/ml; higher concentrations (10¹³)result in expression in some cones, but with adverse retinal effects.Expression in both rods and cones was regulated by 235 bp of the humanIRBP promoter that contains the important cis-acting element identifiedin the mouse proximal promoter. See, al-Ubaidi M R, et al. (1992), JCell Biol 119:1681-1687 and Boatright J H, et al. 1997) Mol Vis 3:15,both incorporated by reference herein. Vectors with this promoter resultin GFP expression in both rods and cones in a dose and time dependentmanner (FIG. S4 of Beltran 2012 which is reproduced as FIG. 8 of USProvisional Patent Application No. 61/670,355). Vector DNA sequenceswere confirmed for accuracy before vector production.

The two-plasmid co-transfection method was used to produce the AAV2/5vector (See, Zolotukhin S, et al. (1999) Gene Ther 6:973-985 which isincorporated by reference herein). Viral particles were titered andresuspended in balanced salt solution (BSS, Alcon Laboratories, FortWorth, Tex.) containing 0.014% Tween-20 at a concentration of 1.5×10¹¹viral vector genomes per mL (vg/ml). Sterility and the lack of endotoxinwere confirmed in the final product.

d. Subretinal Injections and Post Treatment Management

Subretinal injections were performed under general anesthesia aspreviously published (See, e.g., Beltran W A, et al. (2010) Gene Ther.17:1162-1174 and Komaromy A M, et al. (2010) Hum Mol Genet 19:2581-2593,both incorporated by reference herein). The volume injected wasdependent on age/eye size: 70 μl and 150 μl, respectively, at 5 and 28wks of age, with the therapeutic vector injected in the right eye, andBSS injected in the left. At the time of the injection, the location andextent of the subretinal blebs were recorded on fundus photographs orschematic fundus illustrations; in all cases the blebs flattened and theretina reattached within 24 hrs. Failed subretinal injection thatrefluxed into the vitreous was found in one dog that was maintainedthroughout the study to determine potential therapeutic efficacy and/orocular complications by the intravitreal route.

e. Clinical Assessment and In Vivo Retinal Imaging

Ophthalmic examinations were conducted throughout theinjection-termination time interval. En face and retinal cross-sectionalimaging was performed under general anesthesia, and retinalcross-sections with SD-OCT were analyzed as described (Aleman T S, etal. (2007) Invest Ophthalmol Vis Sci 48:4759-4765; Jacobson S G, et al.(2011) Invest Ophthalmol Vis Sci 52:70-79; Jacobson S G, et al. (2009)Invest Ophthalmol Vis Sci 50:1886-1894; Jacobson S G, et al. (2008)Invest Ophthalmol Vis Sci 49:4573-4577, all of which are incorporated byreference herein). En face and retinal cross-sectional imaging wasperformed with the dogs under general anesthesia. Overlapping en faceimages of reflectivity with near-infrared illumination (820 nm) obtained(HRA2 or Spectralis HRA or Spectralis HRA+OCT, Heidelberg, Germany) with30° and 55° diameter lenses to delineate fundus features such as opticnerve, retinal blood vessels, boundaries of injection blebs, retinotomysites and other local changes. Custom programs (MatLab 6.5; TheMathWorks, Natick, Mass.) were used to digitally stitch individualphotos into a retina-wide panorama. In a subset of eyes,short-wavelength (488 nm) illumination was used to delineate theboundary of the tapetum and pigmented RPE. Spectral-domain opticalcoherence tomography (SD-OCT) was performed with linear and raster scans(RTVue-100, Optovue, Inc. Fremont, Calif. or Spectralis HRA+OCT,Heidelberg, Germany).

Linear scans were placed across regions or features of interest such asbleb boundaries in order to obtain highly resolved local retinalstructure. The bulk of the cross-sectional retinal information wasobtained from overlapping raster scans covering large regions of theretina. Either 6×6 mm (101 lines of 513 longitudinal reflectivityprofiles (LRPs) each, no averaging, Optovue) or 9×6 (49 lines of 1536LRPs each, averaging 8-10, Spectralis) was used.

Post-acquisition processing of OCT data was performed with customprograms (MatLab 6.5; The MathWorks, Natick, Mass.). For retina-widetopographic analysis, integrated backscatter intensity of each rasterscan was used to locate its precise location and orientation relative toretinal features visible on the retina wide mosaic formed by NIRreflectance images. Individual LRPs forming all registered raster scanswere allotted to regularly spaced bins (1°×1°) in a rectangularcoordinate system centered at the optic nerve; LRPs in each bin werealigned and averaged. Intraretinal peaks and boundaries corresponding tohistologically definable layers were segmented semi-automatically withmanual override using both intensity and slope information ofbackscatter signal along each LRP. Specifically, the retina-vitreousinterface, OPL, outer limiting membrane (OLM), signal peak near theinner/outer segment (IS/OS) junction, and the RPE were defined. In thesuperior retina of the dog, backscatter from the tapetum forms thehighest intensity peak, and RPE and IS/OS peaks are located vitreal tothe tapetal peak. ONL thickness was defined from the sclerad transitionof the OPL to the OLM, and ONL thickness topography was calculated. Inaddition, the topography of IS/OS backscatter intensity was calculatedby first measuring the mean backscatter intensity within ±8 μm of theIS/OS peak and then normalizing this value by the mean backscatterintensity of the first 75 μm of retina sclerad to the retina-vitreousinterface. For all topographic results, locations of blood vessels,optic nerve head and bleb boundaries were overlaid for reference.Further quantitative comparisons were achieved by sampling the ONLthickness of a 10° wide band along the vertical meridian crossing theoptic nerve head.

f. Electroretinography

Dogs were dark-adapted overnight, premedicated, and anesthetized asdescribed (Acland G M, et al. (2001) Nature Genetics 28:92-95; Kijas JW, et al, (2002) Proc Natl Acad Sci USA 99:6328-6333, both of which areincorporated by reference herein). Pupils were dilated with atropine(1%), tropicamide (1%) and phenylephrine (10%). Pulse rate, oxygensaturation, and temperature were monitored. Full-field ERGs wererecorded with Burian-Allen (Hansen Ophthalmics, Iowa City, Iowa) contactlens electrodes and a computer-based system. White flashes of low-energy(10 μs duration; 0.4 log scot-cd.s.m-2) and high-energy (1 ms duration;3.7 log scot-cd.s.m-2) were used under dark-adapted and light-adapted(1.5 log cd.m-2 at 1 Hz stimulation, 0.8 log cd.m-2 at 29 Hzstimulation) conditions. Leading edges of high-energy flash responseswere measured at the fixed time point of 4 ms (Acland G M, et al. (2005)Mol Ther 12:1072-1082, incorporated by reference herein) to quantifyretinal function dominated by the rod photoreceptors and thepeak-to-peak amplitude of the low-energy flashes at 29 Hz were measuredto quantify retinal function dominated by the cone photoreceptors.Assessment of visual behavior using qualitative or quantitative measureswas not performed in treated animals because, at the disease stagesstudied, both mutant strains (untreated) retain sufficient rod and conevisual function that they are not distinguishable from normal.

g. Tissue Processing and Morphologic Assessment

Dogs were euthanatized by intravenous injection of euthanasia solution(Euthasol, Virbac, Ft. Worth, Tex.), and the globes enucleated, fixedand processed as previously described (Beltran W A et al, (2006) InvestOphthalmol Vis Sci 47:1669-1681, incorporated by reference herein).Serial 10 μm thick retinal cryosections that encompassed treated andnon-treated regions were cut (˜700/retina), and a subset stained withH&E; vascular landmarks were used to place the section on the en facecSLO images and subsequent en face ONL maps. Immunohistochemistry wasdone in adjacent sections with antibodies directed against rod opsin,human cone arrestin (LUMIf; 1/10 000 provided by Cheryl Craft),R/G-opsin, RIBEYE/CtBP2, PKCα, Goα, calbindin, neurofilament (NF200kDa), and GFAP to examine the expression of molecular markers in treatedand non-treated areas (Beltran W A et al (2006), cited above, andBeltran W A et al (2009) Invest Ophthalmol Vis Sci 50:3985-)995, each ofwhich is incorporated by reference herein). As well, an antibodydirected at the C-terminal domain of human RPGR (Khanna H, et al. (2005)J Biol Chem 280:33580-33587, incorporated by reference herein) was usedto identify the therapeutic transgene in treated eyes. Theantigen-antibody complexes were visualized with fluorochrome-labeledsecondary antibodies (Alexa Fluor, 1:200; Molecular Probes, Eugene,Oreg., USA) with DAPI to label cell nuclei, and digital images taken(Spot 4.0 camera, Diagnostic Instruments, Sterling Heights, Mich.), andimported into a graphics program (Photoshop; Adobe, Mountain View,Calif., USA) for display.

Example 2 RPGR ORF15 mutations lead to photorecepto egeneration inhumans and dogs

Topography of photoreceptors can be mapped across the retina of patientswith RPGR-XLRP by measuring the thickness of the outer (photoreceptor)nuclear layer (ONL) using cross-sectional OCT retinal imaging. As shownin FIG. 1A of Beltran Wash., et al, (2012 January) Proc Natl Acad SciUSA, 109(6):2132-7 (Beltran 2012), which is reproduced as FIG. 1A ofU.S. Provisional Patent Application No. 61/670,355, in normal eyes(inset), ONL thickness peaks centrally and declines with distance fromthe fovea. XERP patients with ORF15 mutations can have different diseasepatterns. A common pattern shows dramatic photoreceptor losses withrelatively greater retention of ONL thickness at and near the cone-richfoveal region surrounded by a zone of detectable but markedly thinnedONL (FIG. 1A of Beltran 2012 which is reproduced as FIG. 1A of U.S.Provisional Patent Application No. 61/670,355). RPGR disease expressionalso includes the less common phenotype characterized by loss of centralphotoreceptors and diseased, yet better preserved, peripheralphotoreceptors (FIG. 1A of Beltran 2012 which is reproduced as FIG. 1Aof U.S. Provisional Patent Application No. 61/670,355). The presentexamples, taken together with previous observations, demonstrate therecan be a spectrum of human RPGR-XLRP phenotypes. Most of the phenotypeshave rod more than cone dysfunction by ERG.

The two canine models can also be studied with cross-sectional retinalimaging, such as we used for human patients, and topographicalphotoreceptor maps generated and compared with normal data (FIG. 1B ofBeltran 2012 which is reproduced as FIG. 1B of U.S. Provisional PatentApplication No. 61/670,355). Of translational importance, a spectrum ofdisease patterns also occurs in the canine models. XLPRA1 dogs, forexample, can show ONL thinning with relative preservation of a regionimmediately superior to the optic nerve, corresponding to the highphotoreceptor density of the visual streak. In contrast, an example ofan XLPRA2 photoreceptor map shows a pattern of retina-wide ONL thinning,but more pronounced losses in the central retina, corresponding to thevisual streak, than in peripheral retina.

The natural history of photoreceptor degeneration was determined toselect the age and retinal site for treatment in XLPRA1 and XLPRA2 (FIG.1C of Beltran 2012 which is reproduced as FIG. 1C of U.S. ProvisionalPatent Application No. 61/670,355). Spatio-temporal distribution ofphotoreceptor degeneration and the disease course were determined byquantifying ONL thickness along the vertical meridian (FIG. 1C ofBeltran 2012 which is reproduced as FIG. 1C of U.S. Provisional PatentApplication No. 61/670,355). Wild type dogs (WT) (n=5, ages 7-43 wks)show a relatively uniform ONL thickness with slightly higher values(averaging 57 μm) superior to the optic nerve up to eccentricities of35°, and slightly lower values (averaging 54 μm) inferior to the opticnerve up to 25°, XLPRA1 at younger ages (n=7, ages 7-28 wks) shows ONLthickness that is within or near normal limits (FIG. 1C of Beltran 2012which is reproduced as FIG. 1C of U.S. Provisional Patent ApplicationNo. 61/670,355). Older XLPRA1 (n=6, ages 56-76 wks) shows ONL thinningin the inferior retina and relative preservation of the visual streakregion immediately superior to the optic nerve (FIG. 1C, brackets, ofBeltran 2012 which is reproduced as FIG. 1C of U.S. Provisional PatentApplication No. 61/670,355). There can be greater differences amongolder XLPRA1 eyes, with some results near the lower limit of normal andothers showing substantial ONL loss below 50% of WT (FIG. 1C of Beltran2012 which is reproduced as FIG. 1C of U.S. Provisional PatentApplication No. 61/670,355), consistent with variable severity ofdisease as previously reported.

XLPRA2 at the youngest ages examined (n=2, ages 8, 22 wks), we observedretina-wide ONL thinning that tended to be greater in the central retina(44% of WT), corresponding to the visual streak, than in the periphery(60% of WT) (FIG. 1C of Beltran 2012 which is reproduced as FIG. 1C ofU.S. Provisional Patent Application No. 61/670,355). Older XLPRA2 dogs(n=3, ages 36-59 wks) show more ONL thinning with a tendency for greatercentral and inferior retinal disease (30% of WT) than in the superiorperipheral retina (45% of WT) (FIG. 1C of Beltran 2012 which isreproduced as FIG. 1C of U.S. Provisional Patent Application No.61/670,355). ONL thickness in the oldest XLPRA1 and XLPRA2 eyes wassubstantially reduced (FIG. 1C of Beltran 2012 which is reproduced asFIG. 1C of U.S. Provisional Patent Application No. 61/670,355). Rod andcone retinal function in young and older dogs with XLPRA1 and. XLPRA2was measured by ERG. Both XLPRA1 and XLPRA2 diseases could becharacterized as having more rod than cone dysfunction. Younger XLPRA1eyes (n=6) showed abnormal (4/6) rod function but normal cone function(FIG. 1D of Beltran 2012 which is reproduced as FIG. 1D of U.S.Provisional Patent Application No. 61/670,355) whereas older XLPRA1 eyes(n=7) showed abnormal rods (6/7) and cones (5/7) (FIG. 1D of Beltran2012 which is reproduced as FIG. 1D of U.S. Provisional PatentApplication No. 61/670,355). Younger XLPRA2 eyes (n=3) had abnormal rodfunction but mostly (2/3) normal cone function, but older XLPRA2 eyes(n=6) had abnormal rod and cone function (FIG. 1D of Beltran 2012 whichis reproduced as FIG. 1D of U.S. Provisional Patent Application No.61/670,355). Defining the differences in structural and functionalnatural history of XLPRA1 and XLPRA2 diseases showed a sufficientoverlap in the non-invasive studies in dog and man to validate the useof the dog models in proof-of-concept studies of treatment that may berelevant to RPGR-XLRP patients.

Example 3 Treatment of XLPRA with Gene Knockdown and ReplacementStrategy In Vivo Findings

It was hypothesized that a gene knockdown and replacement strategy wouldbe necessary in order to overcome the effects of the mutated RPGR gene.Thus, a short hairpin RNA was encoded into a construct containing acanine shortened RPGRORF15 cDNA, which has had 708 bp removed in framefrom the repetitive region of ORF15 (cRPGR_(short)). Additional silentmutations were included in the cRPGR sequence to “harden” the sequenceto the siRNA.

Subretinal injection of the cRPGR_(short) cDNA under the control ofhIRBP promoter (AAV2/5-hIRBP-cRPGR_(short)-shRNA5) was performed mXLPRA2 dogs. Treatment was initiated at 5 weeks, after disease onset.Severe retinal lesions of retinal dysplasia were observed at 17 weeksfollowing sub-retinal injection. No rescue was seen (Table 2).

Example 4 Treatment of XLPRA with Gene Augmentation Therapy In VivoFindings

Subretinal injection of the full-length human RPGRORF15 cDNA undercontrol of hIRBP (AAV2/5-hIRBP-hRPGR) (FIG. 1) promoter was performed inboth XERPA1 and XLPRA2, and under control of hGRK1 (AAV2/5-hGRK1-hRPGR)(FIG. 2) promoter in XLPRA2 (Table 1). In XLPA1, treatment was initiatedat 28 wks, before photoreceptor loss, and monitored to 77 wks, wellafter the start of degeneration (FIG. 1C of Beltran 2012 which isreproduced as FIG. 1C of U.S. Provisional Patent Application No.61/670,355). In XLPRA2, the injections were performed at 5 wks of age,and the study terminated at 38 wks. In contrast to the treatmentfailures discussed above, the full length human RPGRORF15 (driven byhIRBP or hGRK1 promoters) was therapeutically effective,

As shown in FIG. S5 of Beltran 2012, which is reproduced as FIG. 9 ofU.S. Provisional Patent Application No. 61/670,355) demonstrates thatAAV2/5 vector with hIRBP promoter targets expression to rods and cones.This figure shows native GFP fluorescence (green) in normal canineretina 2 and 8 wks following subretinal injection of AAV2/5-hIRBP-hGFP.150 μl injections of 2 vector titers were used. GFP fluorescence inphotoreceptors is present by 2 wks (A1, A2), and is increased at 8 wks(B1, B2). Expression is found in both rods and cones as confirmed bycolocalization with cone arrestin in retinas treated with lower vectordoses which result in fewer cones transduced (C). More photoreceptorsare labeled at the higher dose, and expression is sustained during 8week treatment period.

The positive treatment response was detectable in vivo. Treated eyes ofXLPRA1 dogs had thicker ONL in the superior peripheral retina,specifically on the treated side of the subretinal injection area (bleb)boundary compared to the untreated side (FIG. 2A of Beltran 2012 whichis reproduced as FIG. 2A of U.S. Provisional Patent Application No.61/670,355). Additionally, the signal peak corresponding to the regionof photoreceptor inner and outer segments (IS/OS) was more intense andbetter organized on the treated side (FIG. 2A of Beltran 2012 which isreproduced as FIG. 2A of U.S. Provisional Patent Application No.61/670,355). Treated eyes of XLPRA2 dogs showed thicker ONL on thetreated side or higher intensity signal at the level of the IS/OS (FIG.2A of Beltran 2012 which is reproduced as FIG. 2A of U.S. ProvisionalPatent Application No. 61/670,355). To understand better therelationship between the treatment bleb and local retinal structure, ONLthickness was mapped across wide expanses of the treated and controleyes (FIG. 2B of Beltran 2012 which is reproduced as FIG. 2B of U.S.Provisional Patent Application No. 61/670,355). XLPRA1 dog H484 at 76weeks of age had a clearly demarcated zone of ONL retention within thetreatment bleb in superior peripheral retina (FIG. 2B of Beltran 2012which is reproduced as FIG. 2B of U.S. Provisional Patent ApplicationNo. 61/670,355). There was ONL degeneration outside the bleb in thesuperior temporal retina. In the central retinal region where XLPRA1dogs at this age retain near normal ONL thickness (FIG. 1C of Beltran2012 which is reproduced as FIG. 1C of U.S. Provisional PatentApplication No. 61/670,355)), a transition across the bleb boundary wasless detectable (FIG. 2B of Beltran 2012 which is reproduced as FIG. 2Bof U.S. Provisional Patent Application No. 61/670,355)).

XLPRA1 dog H483 with a smaller subretinal bleb had similar findings inthe superior peripheral region with local evidence of ONL thicknessretention inside the bleb boundary. More centrally, both treated anduntreated regions retained near normal ONL thickness and there was nochange in ONL thickness corresponding to the bleb boundary (FIG. 2B ofBeltran 2012 which is reproduced as FIG. 2B of U.S. Provisional PatentApplication No. 61/670,355). XLPRA2 dog Z412 showed a region withpreserved ONL that corresponded to the bleb boundary; ONL was abnormallythinned outside this boundary (FIG. 2B of Beltran 2012 which isreproduced as FIG. 2B of U.S. Provisional Patent Application No.61/670,355). Longitudinal follow-up from 21 to 36 wks showed the timecourse of ONL degeneration outside the bleb of the treated eye and inthe BSS-injected control eye (FIG. S1 of Beltran 2012 which isreproduced as FIG. 5 of U.S. Provisional Patent Application No.61/670,355). XLPFA2 dog Z414 showed a region of slight ONL thicknessretention approximately corresponding to the bleb boundary (Fig.2B ofBeltran 2012 which is reproduced as FIG. 2B of U.S. Provisional PatentApplication No. 61/670,355).

Changes at the level of photoreceptor IS/OS were quantified. Backscatterintensity at this layer was segmented and mapped (FIG. 2C of Beltran2012 which is reproduced as FIG. 2C of U.S. Provisional PatentApplication No. 61/670,355). IS/OS intensity maps of three of thetreated dogs (H484, H483 and Z412) were similar to the ONL maps, suchthat regions of retained ONL corresponded to higher intensity. In thecase of Z414, the treated region showed substantially higher backscatterintensity at the IS/OS layer and this was consistent with the betterlayer definition apparent in individual scans (FIG. 2A of Beltran 2012which is reproduced as FIG. 2A of U.S. Provisional Patent ApplicationNo. 61/670,355). Comparison of the treated and BSS-injected control eyesshowed the clearly delineated retinal regions with treatment-relatedeffects (FIG. 2C, hashed, of Beltran 2012 which is reproduced as FIG. 2Cof U.S. Provisional Patent Application No. 61/670,355). ERGs wereevaluated in terms of interocular asymmetry (FIG. 2D of Beltran 2012which is reproduced as FIG. 2D of U.S. Provisional Patent ApplicationNo. 61/670,355). Signals were larger in the treated eyes of three dogs(H484, Z412 and Z414) for photoreceptor responses dominated by rods, andfor post-receptoral bipolar cell responses mediated by both rods andcones. H483 had the least degenerate retina and normal amplituderesponses bilaterally (FIGS. 2D and S2 of Beltran 2012 which isreproduced as FIGS. 2D and 6 of U.S. Provisional Patent Application No.61/670,355) that were symmetric for cones and asymmetric for rods,favoring the untreated eye.

These data are summarized in Tables 1 and 2 below:

TABLE 1 Genotype Age Morphology/ Animal/ (wks)¹ Agent OCT ERG⁵ IHC InnerhRPGR eye Begin End Injected ONL³ IS/OS⁴ Rod Cone PR⁶ OPL⁷ Retina⁸ Exp⁹XLPRA1 28 77 hIRBP- + + + + N N N +3 H484- hRPGR RE XLPRA1 28 77 BSS −− + + D D D − H484-LE XLPRA1 28 77 hIRBP- + + − − N N N +2 H483- hRPGRRE XLPRA1 28 77 BSS − − − − D D D − H483-LE XLPRA2 5 38 hIRBP- + + + + NN N +2 Z412-RE hRPG XLPRA2 5 38 BSS − − + + D D D − Z412-LE XLPRA2 5 38hGRK1- − + + + P P P +1 Z414-RE hRPGR XLPRA2 5 38 BSS − − + + D D D −Z414-LE XLPRA2 5 38 hGRK1- ND ND ND ND D D D − Z413-RE hRPGR¹⁰ NOTES:BSS—balanced salt solution; RE—right eye; LE—left eye; ND—not done. ¹Thespan of ages (in weeks) from treatment to termination. ²Subretinalinjections with a volume of 70 μl at 5 wks of age, and 150 μl at 28 wks.AAV2/5 vector injections had a titer of 1.5 × 1011 vg/ml. Dog Z413 had70 μl injected into the vitreous and served as control. ³Existence of aregion of retained outer nuclear layer (ONL) within the injection blebcompared to outside the bleb as measured by optical coherence tomography(OCT); + = positive treatment outcome, − = no response to treatment.⁴Existence of a region of higher inner segment/outer segment (IS/OS)reflectivity within the injection bleb compared to outside the bleb asmeasured by OCT; + = positive treatment outcome, − = no response totreatment. ⁵Interocular asymmetry of the rod- or cone-dominatedelectroretinogram (ERG) amplitudes ⁶Photoreceptors (PR). Structure ofrods, cones and outer nuclear layer in treated vs untreated regions, andreversal of rod and cone opsin mislocalization. N = normal, rescue; D =diseased, no rescue; P = partial rescue. ⁷Outer plexiform layer (OPL),Pre- and post-synaptic terminal structures, including presence of normalelongated bipolar dendrites as determined by immunohistochemistry (IHC)using antibodies that label photoreceptor synaptic terminals and bipolarcells. N = normal, rescue; D = diseased, no rescue; P = partial rescue.⁸Reversal/prevention of inner retinal remodeling. N = normal, rescue; D= diseased, no rescue; P = partial rescue. ⁹hRPGR expression in treatedarea determined with a C-terminal antibody. Labeling limited to rods andcones and graded as − (no label), +1 (weak), +2 (moderate), and +3(intense). ¹⁰Represents an intra vitreal control.

TABLE 2 Age (wks) Promoter- Vector Animal/ Begin/ transgene titerOutcome Process End (# of eyes) (vg/ml) Rescue Complications XLPRAl/26-28/ mOP-cRPGR (1) 1.5 × 10¹¹ No Multifocal rosettes (1) Augmentation31-37 hlRBP-HiscRPGR (4) 1.5 × 10¹¹ No Multifocal rosettes (4)hGRK1-hRPGR (1) 1.5 × 10¹¹ ** Small retinal detachments (1) XLPRA2/5-22/ CBA-GFP-H1- 2.8- No None Knockdown 20-39 siRNA3 (1) 2.9 × 10¹¹CBA-GFP-H1- No None siRNA5 (1) CBA-GFP-H1- No None siRNA5 (1)CBA-GFP-H1- No None siRNA5 (2) XLPRA2/ 5/22 hlRBP-cRPGR_HT- 1.5 × 10¹⁰-No Multifocal rosettes (2) Knockdown + H1-siRNA5 (3) 1.5 × 10¹¹ None (1)Augmentation hlRB-cRPGR_HT- 1.5 × 10¹⁰- No None (2) H1-siRNA5 (2)-IV 1.5× 10¹¹ NOTES: c = canine; h = human; IV—intravitreal control; mOP =minimal opsin promoter; CBA = chicken beta actin promoter⁶⁵. ** Mild andnon-uniform hRPGR expression in treatment area, partial recovery ofopsin mislocalization. Photoreceptor rescue was not interpretablebecause of the retinal detachments, and early termination secondary tothese complications.

Previous hypotheses about XLPRA2 being due to a toxic gain of function⁶³led to attempts to downregulate mutant RPGR expression in order toimprove the disease. Two KD reagents, shRNA3 and shRNA 5, were used thatwere effective downregulating canine RPGR expression in vitro but therewas no rescue. Simultaneous replacement of RPGR used a single viralconstruct that combined shRNA5 and a resistant abbreviated⁶⁴ canine RPGRcDNA that had a 5′ 6×His tag. Subretinal treatment with high vectortiter resulted in no efficacy and retinal toxicity. Similar retinaltoxicity was observed by augmentation alone with the abbreviated canineRPGR cDNA without the His tag.

Example 5 Gene Augmentation Rescues Photoreceptors and ReversesMislocalization of Rod and Cone Opsins in Both XLPRA Genotypes

Assessment of retinal morphology in tissue sections that included thebleb boundary confirmed the in vivo imaging results of retention of ONLthickness and photoreceptor preservation in subretinally-treated areas(Panels 1-5 in FIGS. 3 and S3 of Beltran 2012 which are reproduced asFIGS. 3 and 7 of U.S. Provisional Patent Application No. 61/670,355).Intravitreal vector administration was comparable to no treatment (Table1). In the three dogs treated with AAV2/5-hIRBP-hRPGR (H484, H483, Z412)rod and cone IS and OS structure was normal within the bleb boundary. Inthe untreated areas, IS were short and OS sparse and irregular (Panels3, 4 in FIGS. 3 and S3 of Beltran 2012 which are reproduced as FIGS. 3and 7 of U.S. Provisional Patent Application No. 61/670,355). In Z414,treated with AAV2/5-hGRK1-hRPGR, a milder yet positive photoreceptorrescue was observed in the bleb area (FIG. S3C of Beltran 2012 which isreproduced as FIG. 7C of U.S. Provisional Patent Application No.61/670,355). Immunolabeling with an antibody directed against humanRPGRORF15 detected robust hRPGR protein expression limited tophotoreceptors in the treatment area (Table 1). Labeling was foundthroughout the IS and synaptic terminals in the four dogs, as well asthe rod and cone perinuclear region of H484 (Panels 6-8 in FIGS. 3 andS3 of Beltran 2012 which are reproduced as FIGS. 3 and 7 of U.S.Provisional Patent Application No. 61/670,355). Finally, themislocalization of rod and cone opsins, a feature of the disease inhuman, mouse and dog, was reversed. (Panels 9,10,12,13 in FIGS. 3 and S3of Beltran 2012 which are reproduced as FIGS. 3 and 7 of U.S.Provisional Patent Application No. 61/670,355) in the three dogs treatedwith AAV2/5-hIRBP-hRPGR. Reduced yet distinct rod and R/G cone opsinmislocalization was apparent in Z414, treated with AAV2/5-hGRK1-hRPGR(FIG. S3C of Beltran 2012 which is reproduced as FIG. 7C of U.S.Provisional Patent Application No. 61/670,355).

Example 6 Prevention of Secondary OPL, Bipolar Cell and Inner RetinalDisease

In XLPRA, like other primary photoreceptor diseases, OPL and innerretinal abnormalities are common secondary effects. In untreatedregions, narrowing of the OPL was associated with compressedphotoreceptor synaptic terminals (Panels 2, 5 in FIGS. 3 and S3 ofBeltran 2012 which are reproduced as FIGS. 3 and 7 of U.S. ProvisionalPatent Application No. 61/670,355), and a reduction of the number ofCABP2-labeled synaptic ribbons in rod and cone terminals (Panels 1, 2 inFIGS. 4 and S4 of Beltran 2012 which are reproduced as FIGS. 4 and 8 ofU.S. Provisional Patent Application No. 61/670,355). In parallel, rodand cone bipolar cell dendrites retracted (Panels 3, 4 in FIGS. 4 and S4of Beltran 2012 which are reproduced as FIGS. 4 and 8 of U.S.Provisional Patent Application No. 61/670,355). These secondary changeswere absent in treated areas, resulting in a preserved OPL. In contrast,calbindin labeling of horizontal and amacrine cells (Panels 5, 6 inFIGS. 4 and S4 of Beltran 2012 which are reproduced as FIGS. 4 and 8 ofU.S. Provisional Patent Application No. 61/670,355), and their lateralprocesses, was normal and unchanged between treated and untreatedregions. These hallmarks of late stage retinal remodeling in XLPRA werenot expected to be present at the age when dogs were terminated.

The dendritic terminals of horizontal cells, as well as that of ganglioncells, and the nerve fiber layer of treated and untreated regionsappeared normal when labeled with an antibody directed against theneurofilament heavy chain (NF 200 kDa). However, there was punctateNF200 staining in the ONL. Overexpression of neurofilaments is acharacteristic of axonal injury in several neurodegenerative disorders,and occurs in this and other retinal diseases. This finding wasrestricted to the untreated regions of all dogs, and was absent orreduced in treated areas (Panels 5, 6 in FIGS. 4 and S4 of Beltran 2012which are reproduced as FIGS. 4 and 8 of U.S. Provisional PatentApplication No. 61/670,355). GFAP immunolabeling clearly delineateduntreated regions that showed increased Müller glia reactivity, whereasit diminished in the transition zone between treated and untreatedregions, and was absent in the bleb area (Panels 7, 8 in FIGS. 4 and S4of Beltran 2012 which are reproduced as FIGS. 4 and 8 of U.S.Provisional Patent Application No. 61/670,355). In summary, innerretinal rescue was complete in 3 of 4 treated eyes; rescue was partialfor the one treated with AAV2/5-hGRK1-hRPGR where rod neurite sproutingextended into the inner retina (Table 1), and NF200 labeling pattern wasintermediate between normal and disease (FIG. S4D of Beltran 2012 whichis reproduced as FIG. 8D of U.S. Provisional Patent Application No.61/670,355). The results clearly show that targeting RPGR augmentationto photoreceptors in both XLPPA1 and XLPRA2 corrects the primaryphotoreceptor defect, and has beneficial downstream effects OPL andinner retinal abnormalities are prevented or reversed.

Example 7 Discussion

Recent successes using gene replacement to treat LCA2, the autosomalrecessive RPE disease due to RPE65 mutations, have paved the way forconsidering gene therapy for treating other incurable humanretinopathies. XLRP is among candidate diseases for treatment because itcan be identified in the clinic, either through pedigree analysis,carrier identification or by the fact that there is a high frequency ofXLRP among simplex males with RP; and mutations in RPGRORF15 account forabout 75% of XLRP patients. The current results showing treatmentefficacy in two large animal models of human RPGRORF15-XLRP stronglysuggest that a gene augmentation strategy is a viable option for thisphotoreceptor ciliopathy, and complements successful rod rescue in amurine model of the Bardet-Biedl syndrome ciliopathy.

The disease in humans and in animal models is not, however, withoutcomplexity and future therapy of the human disease will need to beapproached with caution. For example, there are modifiers that mayaffect disease expression in both patients and dog models, and there isa spectrum of phenotypes between and within RPGRXLRP families and in thedog XLPRA1 model. The phenotypic diversity may be a potential obstacleto patient selection, and points to the need for more than a moleculardiagnosis and patient's age as criteria to determine candidacy fortreatment. In support of genotype data there must be complementingdetailed non-invasive retinal imaging and function studies. Thetemptation should be resisted in early human treatment approaches to tryto design a treatment to fit all phenotypes and all disease stages. Thedog diseases are mainly rod>cone degenerations, and there was efficacyin treating both the severe XLPRA2 with central retinal degeneration,and the less severe XLPRA1 with central retinal preservation usingvectors that targeted both rods and cones. Not included in the caninedisease spectrum, however, are certain human RPGR-XLRP phenotypes, suchas mild cone>rod or cone dystrophies. Some patients can show verylimited or even normal rod function, and cone targeting strategies mustbe developed for these subtypes. Proof-of-principle studies targetingcone diseases already have been successful in both mouse and dog modelswith mutations in cone phototransduction or cyclic GMP channel genes, sotranslation to the clinic would be expedited.

The reported intrafamilial variation of phenotypes neither excludes norincludes entire pedigrees from participation, but further strengthensthe case for complete clarification of phenotype in individual patients.Furthermore, in the present study, there was no attempt to target thevery central retina; the extracentral subretinal approach as used in thedogs would be the advisable strategy for early phase human clinicaltrials based on the current observations. However, many RPGR patientsshow continued survival of foveal cones and impaired but useful visualacuity in late disease stages. As subfoveal injections of vector-genehave been shown to cause loss of diseased foveal cones, alternate meansof therapeutic gene delivery should be considered. Advances inintravitreal delivery systems to treat the outer retina, e.g. usingmutant AAV capsid vectors, eventually could allay the safety concerns intreating residual foveal cones.

While it is clear that RPGR-associated disease is common and generallysevere, the function of the gene, and the association between mutationand disease are less well understood. RPGR has a complex splicingpattern with multiple tissue-/cell-specific isoforms, is known tointeract with a number of ciliary proteins, acts as a gunaninenucleotide exchange factor for small GTPase RAB8A and may have a role invertebrate development. Such complexity may account partially for thevariability in disease phenotype. In general, loss of function or gainof function mechanisms have been proposed, a suggestion that each wouldrequire different therapeutic approaches. Although our present studiescannot rule out either mechanism as causal to disease, the resultsclearly indicate that gene augmentation alone is effective in preventingdisease, or arresting progression of and reversing the degenerativeprocess in canine models of ORF15 mutations. These fundamental findingsallow us to move forward therapeutically towards translational studieswhile the specific disease mechanisms await further elucidation.

Our results emphasize that targeting therapy to rod and conephotoreceptors is essential for functional and structural rescue inRPGR-associated retinal disease. The hIRBP promoter that regulatesexpression of the therapeutic gene results in robust expression ofreporter or therapeutic genes in both cell types (FIG. S5 and Panels 6-8in FIGS. 3 and S3 of Beltran 2012 which are reproduced as FIGS. 9, 2 and7, respectively, of U.S. Provisional Patent Application No. 61/670,355),and expression is sustained. As IRBP also is expressed in human cones,we expect efficient targeting of rods and cones with this promoter infuture translational studies. When regulated by the hGRK1 promoter, thetherapeutic transgene expression was low in rods, and to a lesser extentin cones. Remaining photoreceptor structure, albeit abnormal, wasconsiderably improved over untreated regions. However, because GRK1 isexpressed in human cones, we expect targeting efficiency of GRK to beincreased in humans. See, e.g., Weiss, E. R. et al, 2001. J Neurosei21(23): 9175-9184, which is hereby incorporated by reference.

In XLPRA1, treatment before disease onset prevented disease development.Further, treatment of XLPRA2 after disease onset, and whilstphotoreceptor cell death was ongoing (at 5 wks cell death is ˜50% of themaximal rate determined by TUNEL labeling), arrested progression of thedisease, and the morphology of the remaining photoreceptors was restoredto normal. At least for the stages of disease studied, this therapeuticvector was highly effective, and warrants further studies fortranslational applications. In both models, treatment with thehIRPB-hRPGR therapeutic vector prevented (XLPRA1) or reversed (XLPRA2)rod and R/G cone opsin mislocalization, a feature of the disease inhuman, mouse and dog, and a putative early marker of photoreceptor celldeath.

A characteristic feature of photoreceptor degenerations is progressivechanges in the OPL, bipolar cells and inner retinal layers. These werewidespread in untreated areas, but reversed to normal in treated areas,particularly when the AAV2/5-hIRPB-hRPGR vector was used. Prevention ofremodeling occurred when treating XLPRA1 retinas prior to disease onset,while in XLPRA2, early OPL synaptic changes, bipolar cell abnormalities,and inner retinal abnormalities were abrogated with treatment, andnormal structure ensued. Thus treatment of the primary photoreceptordefect has beneficial downstream effects as OPL and inner retinalabnormalities are prevented or reversed. This may account for theimproved post-receptoral responses recorded from 3 of the 4 treateddogs. Future studies should extend the post-treatment follow-up periodto older ages when degeneration of untreated regions would allow testingof treatment consequences at the visual brain such as with the use ofpupillometry and visual evoked potentials, and ultimately with visualbehavior. Subretinal treatment in XLPRA canine models of RPGRORF15-XLRPwith AAV2/5 vectors and the full length human RPGRORF15 cDNA waseffective in preserving photoreceptor structure and function.

Example 8 Corrective Gene Therapy for RPGR-XLRP Rescues Canine Model atMid-Stage Disease

The inventors next investigated whether gene therapy delivered at a moreadvanced stage of disease can still provide a positive outcome. AnAAV2/5 vector construct (titer: 1.51×10 vg/ml) carrying full-lengthhuman RPGRORF15 cDNA under the control of a hIRBP promoter was injectedsubretinally in three 12-wk-old XLPRA2 dogs. At that age, there ison-going cell death and the ONL thickness is reduced by ˜40%. Inaddition, one XLPRA2 dog was injected shortly after the onset of disease(5.1 wks of aac) as an early disease stage control. Contra-lateral eyeswere either injected with BSS, or received a similar dose of viralconstruct intravitreally. Photoreceptor structure and function wasassessed by means of non-invasive retinal imaging (cSLO/SD-OCT) and ERGat 39 and 42 weeks of age, respectively.

In vivo retinal imaging showed preserved ONE thickness in the treatedretinal areas. Rod and cone ERG function was greater in treated than incontrol eyes. Both ONL thickness and ERG responses were better preservedin the animal treated at 5.1 weeks than in the 3 dogs injected at 12weeks of age.

These results show that a sustained and beneficial effect onphotoreceptor structure and retinal function can be achieved even whendelivering RPGR acne augmentation at a mid-stage of XLRP disease. Thishas important translational application given that patients are likelyto have more advanced disease at the time of treatment.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and illustrativeexamples, make and utilize the compositions of the present invention andpractice the claimed methods. While the invention has been described andillustrated herein by references to various specific materials,procedures and examples, it is understood that the invention is notrestricted to the particular combinations of material and proceduresselected for that purpose. Numerous variations of such details can beimplied as will be appreciated by those skilled in the art.

The disclosures of Beltran W A, et al, (2012 January) Proc Natl Acad SciUSA, 109(6):2132-7 and U.S. Provisional Patent Application No.61/670,355, as well as all patents, patent applications and otherreferences, including the Sequence Listing cited in this specificationare hereby incorporated by reference in their entirety.

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1. A method of preventing, arresting progression of or amelioratingvision loss associated with retinitis pigmentosa in a subject, saidmethod comprising administering to said subject an effectiveconcentration of a composition comprising a recombinant adeno-associatedvirus (AAV) carrying a nucleic acid sequence encoding a normal RPGRgene, or fragment thereof, under the control of regulatory sequenceswhich express the product of said gene in the photoreceptor cells ofsaid subject, and a pharmaceutically acceptable carrier.
 2. (canceled)3. A method of improving photoreceptor structure in a subject in needthereof, said method comprising administering to said subject aneffective concentration of a composition comprising a recombinantadeno-associated virus (AAV) carrying a nucleic acid sequence encoding anormal RPGR gene, or fragment thereof, under the control of regulatorysequences which express the product of said gene in said photoreceptorcells, and a pharmaceutically acceptable carrier. 4-11. (canceled) 12.The method according to claim 1, wherein the composition is administeredby subretinal injection.
 13. The method according to claim 1, whereinthe nucleic acid sequence encodes RPGR exon 1 through ORF15.
 14. Themethod according to claim 1, wherein the nucleic acid sequence isencoded by SEQ ID NO:
 1. 15. The method according to claim 1, whereinthe regulatory sequence comprises the IRBP promoter or GRK1 promoter.16-17. (canceled)
 18. The method according to claim 1, wherein theretinitis pigmentosa is an X-linked form.
 19. The method according toclaim 1, wherein the AAV is a pseudotyped AAV.
 20. The method accordingto claim 19, wherein the AAV is an AAV2/5 pseudotyped AAV. 21-33.(canceled)
 34. The method according to claim 1, wherein the compositionis administered in combination with a secondary therapy.
 35. The methodaccording to claim 34, wherein the secondary therapy is CiliaryNeurotrophic Factor (CNTF).
 36. The method according to claim 1, whereinadministration of the composition is repeated at least once in the sameeye, contralateral eye, or both.
 37. A method of treating or preventingX-linked retinitis pigmentosa (XLRP) in a subject in need thereofcomprising: (a) identifying subject having, or at risk of developing,XLRP; (b) performing genotypic analysis and identifying a mutation inthe retinitis pigmentosa GTPase regulator (RPGR) gene; (c) performingnon-invasive retinal imaging and functional studies and identifyingareas of retained photoreceptors that could be targeted for therapy; (d)administering to said subject an effective concentration of the AAV ofclaim 40, wherein said XLRP is prevented, arrested or ameliorated. 38.The method according to claim 37, wherein the subject is selected from ahemizygous male, a homozygous female and a heterozygous female.
 39. Themethod according to claim 1, wherein the composition comprises arecombinant AAV2/5 pseudotyped adeno-associated virus, carrying anucleic acid sequence encoding a normal RPGR gene, or fragment thereof,under the control of an IRBP promoter which directs expression of theproduct of said gene in said ocular cells, formulated with a carrier andadditional components suitable for subretinal injection.
 40. Arecombinant adeno-associated virus (AAV) carrying a nucleic acidsequence encoding a normal RPGR gene, or fragment thereof, under thecontrol of regulatory sequences which express the product of said genein the photoreceptor cells of said subject.
 41. The AAV according toclaim 40, wherein the nucleic acid sequence encodes RPGR exon 1 throughORF15.
 42. The AAV according to claim 40, wherein the nucleic acidsequence is encoded by SEQ ID NO:
 1. 43. The AAV according to claim 40,wherein the regulatory sequence comprises the IRBP promoter or GRK1promoter. 44-45. (canceled)
 46. The AAV according to claim 40, whereinthe AAV is an AAV2/5 pseudotyped AAV.
 47. (canceled)